1. INTRODUCTION

Lymphomas, the malignant transformation of the cells of the immune system, are broadly classified as Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphoma (NHL). Hodgkin’s lymphoma in most of the cases is known to involve altered B-cells [1]. Globally, in males and females, NHL is ranked the 10^th and 12^th most frequently occurring type of cancers, respectively. It involves the B-lymphocytes and a smaller portion of T-lymphocytes and natural killer cells. However, its classification is not easy as it requires a deep insight into the clinical features and genetic abnormalities [2]. With advances in areas of science such as genomics and molecular biology, chemotherapy and immunotherapy are promising in combating cancer; however, multidrug resistance and drug induced toxicity remains a major cause of concern. Plant-derived drugs, an alternative treatment to fight cancer, are the need of the hour as it has the potential to deliver highly efficient and non-toxic treatment [3]. The efficacy of anticancer agents depends on its ability to increase apoptosis, decrease cell proliferation, induce cell differentiation, modulate intracellular pathways, and inhibit activity of DNA topoisomerase and angiogenesis. Several plants such as Luffa aegyptiaca, Beta vulgaris, Capsicum frutescens, Solenostemma argel, and Colocasia antiquorum are known to have anticancer potentials against acute myeloid leukemia and lymphocyte leukemia [4]. Tragia involucrata Linn. belongs to the Family Euphorbiaceae and is widely distributed in South Asian countries. The various parts of this plant have tremendous ethnopharmacological importance, as it has been known to be used against inflammation, allergy, epilepsy, renal stones, asthma, bronchitis, vomiting, diarrhea, and numerous other ailments [5]. The present study aims to evaluate the apoptotic and tumor reduction potentials of T. involucrata ethanolic extract using lymphoma (DLA solid tumor) models.

2. MATERIALS AND METHODS

2.3. Trypan Blue Exclusion Assay

The use of MTT assay was avoided in our study, as the plant extract contains diverse group of chemicals, and they may reduce the activity of MTT compound, giving false positive results for cell viability [6]. Cytotoxicity assay was conducted according to Strober (2015) [7] with minor modifications. Cells were seeded in six well plates with RPMI medium (1.0 × 10⁵ cells/well) and treated with different concentrations of extract at 37°C in a CO_{2 (}5%) incubator. Trypan blue was added after the incubation and it was kept at room temperature for 3 min. The observations were made in a hemocytometer under a microscope (×20). The percentage was calculated using the formula:

2. 6. DLA-Induced Solid Tumor Model

The nontoxic doses were calculated after conducting acute and subacute toxicity studies. The doses for subacute toxicity studies were calculated as 1/5 (high dose) and 1/10 (low dose) of the nontoxic single oral dose (2g/kg b. wt) given in the acute toxicity studies. The subacute toxicity studies were then conducted with the low and high doses of 200 and 400 mg/kg b. Wt, respectively, for 28 days. After ensuring the safety of these doses, further antitumor studies were conducted using same dosage. Male Swiss albino mice (6–8 weeks old) weighing 25–30 g were sought into five groups (n = 6) for different treatments as follows. Group 1 and 2: Control (untreated tumor induced mice) and vehicle control (1% propylene glycol), respectively, Group 3: Standard (cyclophosphamide drug 10 mg/kg b. wt), and Group 4 and 5: TME low dose (200 mg/kg b. wt) and high dose (400 mg/kg b. wt), respectively. DLA cells were aspirated from the peritoneal cavity of tumor bearing mice and washed in PBS. The tumor was induced by intramuscularly injecting approximately 1 × 10⁶ of DLA cells in 100 μL of saline into the right hind limb of all animals. Twenty-four hours post-tumor cell inoculation, the treatment was initiated by oral administration of the extract and it continued for 10 consecutive days. The tumor growth was measured with a Vernier caliper, by measuring the diameter of its growth in two perpendicular planes. With an interval of 3 days between each reading, the readings were taken for 30 days. The tumor volume was calculated using the formula;

where,

r₁; minor radius and r₂; major radius.

3. RESULTS

3.1. GC–MS Analysis

Identification and characterization of several compounds were possible by comparing the mass spectrum obtained for each with the reference spectrum. The major chemical compounds in ethanol extract identified are listed in Table 1. Figure 1 shows the peaks obtained in the GC–MS analysis.

Table 1: Compounds identified by GC-MS.

Peak no	Retention time	Area (%)	Name	Molecular formula
1	10.308	1.06	Phenylacetaldehyde- diethylacetal	C12H18O2
2	16.41	6.61	Neophytadiene	C20H38
3	17.313	1.29	(E)-Phytol	C20H40O
4	19.447	7.36	Ethyl palmitate	C18H36O2
5	22.297	8.18	Phytol	C20H40O
6	23.358	4.94	Ethyl linolate	C20H26O2
7	23.468	3.18	Ethyl elaidate	C20H38O2
8	23.517	3.80	Linolenic acid	C18H30O2
9	24.010	1.95	Ethyl octadecanoate	C20H40O2
10	30.732	13.08	2-Ethylhexyl phthalate	C24H38O4
11	34.974	13.91	Squalene	C30H50
12	40.139	11.21	Vitamin E	C29H50O2
13	44.720	21.05	Clionasterol	C29H50O2
14	47.183	4.41	Viminalol	C30H50O

Figure 1: Graph showing peaks in GC experiment. [Click here to view]

3.2. LC–MS Analysis

Ten most abundant compounds were identified and the details are given in Table 2. The total ion chromatogram of TME in both positive and negative ion modes is shown in Figure 2.

Table 2: Compounds identified by LC-MS.

Retention time	Name	Molecular Formula	m/z value
4.310	Agathisflavone	C30H18O10	537.082
4.153	Loquatoside	C20H22O11	437.107
3.970	Leufolin A	C30H28O12	579.150
3.813	Quercetin	C15H10O7	303.049
3.805	Echinacin	C30H26O12	577.133
3.257	Apigetrin	C21H20O10	431.097
3.057	Cynaroside	C21H20O11	447.092
2.926	1,2,36-tetrakis-O- galloyl-B-D-glucose	C34H28O22	787.095
2.883	Isoquercetin	C21H20O12	465.104
2.674	Corilagin	C27H22O18	633.072

Figure 2: LC-MS analysis of the extract. (a) Total ion chromatogram of TME in the positive ion mode. (b) Total ion chromatogram of TME in the negative ion mode. [Click here to view]

3.3. Antiproliferation Effect of TME In Vitro

A cell proliferation assay was conducted to examine the growth inhibition exerted by TME on YAC-1 cells. The cells were treated for 12, 24, and 48 h, with different concentrations (5, 10, and 15 μg/mL). The potential of TME to induce cell death for a prolonged time at physiological temperature was revealed, and also, there were a concentration and time dependent reduction in the number of live cells [Figure 3].

Figure 3: Cell proliferation. Graph showing the number of live cells (YAC-1) at three different time points (12, 24, and 48 h) after treatment with extract (5, 10, and 15 μg/ml). Comparisons were made between vehicle controls with the treated groups separately at each time point. The symbol (*) represents statistical significance at P ≤ 0.05. [Click here to view]

3.4. Antitumor Studies

The extract showed that no signs of toxicity in animals as no mortality or behavioral changes were observed. It also reduces cell proliferation in culture, which prompted us to evaluate its tumor reduction potential in the in vivo system. The tumor volume showed significant reduction (P ≤ 0.05) when compared to the vehicle control at the 30^th day of tumor study [Figure 4]. Tumour tissue was further used for histopathological analysis that also showed correlating results [Figure S1, S2].

Figure 4: Tumor volume. Graph showing the volume of DLA solid tumors in Swiss albino mice for 30 days. Untreated control, vehicle control, and standard (Cyclophosphamide) groups are included. Comparisons were made between vehicle control with the extract treated groups separately. The symbol (*) represents statistical significance at P ≤ 0.05. [Click here to view]

4. DISCUSSION

Since aerial parts of T. involucrata were known to be traditionally used in few villages of Kerala, for treating tumors. A study was conducted earlier in ascites tumor models using ethyl acetate and hexane extracts of aerial parts of T. involucrata and a notable decrease in tumor burden and considerable increase in lifespan was observed [8]. The ethyl acetate and chloroform extracts of leaves of T. involucrata showed potent antiproliferative effect when tested against K562 (chronic myelogenous leukemia) cell lines [9]. Further, exploration of this activity was not conducted, and hence, here, we used the ethanol extract (TME) of T. involucrata to conduct an elaborate antitumor study. TME was effectively interfering with cell proliferation and has the ability to induce apoptosis in tumor cells.

The antiproliferative and proapoptotic changes in the tumor tissue can be attributed to several of the phytocompounds present in the extract that was revealed by GC-MS analysis. Neophytadiene detected in TME extracts are known to inhibit the nuclear factor kappa B (NF-κB) translocation to the nucleus and, thereby, significantly reduce the pro-inflammatory cytokine production [10]. In several types of cancer, especially, the ones with hematopoietic origin are known to have mutations in members of NF-κB protein family. Elevated NF-κB levels in tumor tissues promote increased accumulation of proinflammatory cytokines and this creates a protumorigenic microenvironment. Apart from this, the inhibition of NF-κB had inhibitory effects on tumor angiogenesis as it abolishes vascular endothelial growth factor production, a vital angiogenic factor [11]. Phytols detected in the extracts have shown antiproliferative effects and also induced apoptosis in lung cancer cell lines [12]. In HepG2 hepatocellular carcinoma cells, phytols that induced apoptosis were found to induce it through caspase 9/3 activation and also were found to inhibit EMT (epithelial mesenchymal transition) which plays a key role in tumor invasion [13]. Ethyl palmitate and ethyl linoleate present in the extract were also known to have inhibitory effects on NF-κB [14,15]. The NF-κB inhibition by linolenic acid is also well studied and was known to decrease the NO production in both breast and cervical cancer cell lines [16,17]. The inhibition of NF-κB/Rel induced apoptosis in murine B-cells [18]. The silencing of C1s (a protein of the complement system) reduced cell proliferation and viability in ccRcc (clear cell renal carcinoma) [19]. Clionasterol, a compound detected in the TME extract, was known to be a potent inhibitor of C1 protein [20]. Thus, it may play a major role in reducing cell proliferation. Viminalol was also known to possess anti leukemia properties [21].

The LC–MS analysis of the extract revealed several non-volatile compounds present in the extract with pharmacological importance. Most of the compounds detected through LC–MS belong to the flavonoid group of polyphenolic compounds with numerous potentials. Flavonoids are a well-known class of polyphenolic compounds, with immense potential to put a check on cell proliferation and invasiveness, induce apoptosis, arrest cell cycle, and modulate ROS-scavenging enzyme activities [22]. Agathisflavaone showed promising therapeutic potentials in docking studies conducted against important biomacromolecules with therapeutic importance. NF-κB is involved in cell survival and maintenance, and hence, inhibition of NF-κB signaling will lead to apoptosis of the cell. Agathisflavaone showed successful binding to NF-κB as well as cytokines IL-β and IL-10, in which the former is responsible for tumor invasion and metastasis and the latter for inhibiting the immune clearance of tumor cells. Therefore, agathisflavone can be considered a potential source of natural anticancer agent [23]. Quercetin, an important anticancer agent, has therapeutic effects in various types of cancer cells, with various target points. In lymphoma, it has a therapeutic role as they target the mitochondrial system, which is impaired in them. TNF-related apoptosis inducing ligand (TRAIL) is a cytokine that induces apoptosis by binding to its death inducing receptors TRAIL 1 and TRAIL 2. In resistant B-cell lymphoma cell lines, the TRAIL apoptosis mechanism was restored by quercetin. It also inhibited the expression of survivin (BIRC5) and myeloid cell leukemia 1 (Mcl-1), which would otherwise inhibit caspase 9/3. The inhibition of survivin by quercetin aided in caspase 9/3 activation and led to apoptosis. Similarly, the Mcl-1 proteasomal degradation resulted in the activation of BAX, thereby releasing cytochrome c from the mitochondria ultimately leading to apoptosis [24]. Apigetrin showed significant anticancer activity against gastric cancer cells (ASG), by inducing apoptosis through the extrinsic pathway, as the expression of FasL and death receptor 4 proteins were significantly elevated. This eventually led to increase in the caspase 3 and 8 resulting in Poly (ADP-ribose) polymerase cleavage and apoptosis [25]. Apigetrin also induced apoptosis in gastric cancer cells, through the intrinsic pathway by generating ROS, and changing the membrane potential of mitochondria. It was also noted to reduce the IL-6 mediated phosphorylation of signal transducer and activator of transduction (STAT) 3, which could augment the anti-apoptotic response. The upstream signal molecules Janus Kinase-2 and src of STAT 3 signaling pathway were also inactivated by apigetrin, thus exhibiting its strong apoptosis inducing potential [26]. A vital oncogenic determinant called Mesenchymal-Epithelial-Transition (MET) protein (the opposite of EMT) is known to regulate the occurrence, development and deterioration of tumors, and hence decrease in levels of MET will aid in inhibiting the activation of its downstream related pathway thus inhibiting cell survival. Cynaroside was known to enhance the ubiquitin degradation of MET in gastric cancer cells, thus proving to be a good anticancer agent [27]. Corilagin is a very potent anticancer molecule and targets various pathways in various types of cancers. Pheochromocytoma is a hormone secreting tumor of adrenal medulla. Corilagin was found to reduce NF-κB activation in this type of cancer. The NF-κB1 transcription factor has five members namely; Rel A (p65), Rel B, c-Rel, NF-κB1(p50), and NF-B2 (p52). It prevented the phosphorylation of RelA in a concentration dependent manner putting a check on NF-κB activation. In ovarian cancer cells, corilagin was found to interfere with the TGF-β signaling pathway. TGF-β was down regulated by corilagin and it thereby prevented the phosphorylation of its downstream signaling proteins such as suppressor of mothers against decapentaplegic 2, AK strain transforming, and extracellular regulated kinase, leading to cell cycle arrest and apoptosis. In cholangiocarcinoma, corilagin suppressed the expression of downstream target molecules of notch signaling pathway and thus led to apoptosis of the cell [28].

5. CONCLUSION

T. involucrata Linn. is an important ingredient in major Ayurvedic formulations called “Vidaryadi Kwatham Chuna” and “Vidyaryadi Ghrita” [29]. Several potent bioactive molecules were detected in the ethanol extract of T. involucrata. Many of these molecules have strong anticancer properties, and thus, the tumor reduction ability of the plant extract may be attributed to these biomolecules. The biomolecules may be individually contributing to the tumor reduction property or may be functioning in a synergistic manner. Thus, further studies are required to understand the exact molecules which impart the tumor reducing ability.

6. AUTHORS’ CONTRIBUTIONS

All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be an author as per the international committee of medical journal editors (ICMJE) requirements/guidelines.

7. FUNDING

There is no funding to report.

8. CONFLICTS OF INTEREST

The authors declare no conflicts of interest with this manuscript.